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1.
The structure and phospholipid-binding properties of human plasma high density apolipoprotein A-I (apoA-I) has been studied at pH 7.4 and 3.1 by microcalorimetry, circular dichroism and density gradient ultracentrifugation. At pH values of 7.4 and 3.1, apoA-I binds to dimyristoyl phosphatidylcholine (DMPC) to form complexes of similar composition (molar ratio of DMPC/apoA-I of 100) and helical content (67%). At pH 7.4, the lipid-protein association is accompanied by an increase in helical content from 58 to 67% and an exothermic enthalpy of binding (deltaHB) of -90 kcal/mol apoA-I. At pH 3.1, the helical content of apoA-I is increased from 48 to 67% on binding to DMPC and the enthalpy of binding was -170 kcal/mol. We suggest that the difference in the enthalpies of binding (-80 kcal/mol) at pH 3.1 compared to 7.4 is due to the greater coil leads to helix transition at the lower pH.  相似文献   

2.
Analytical isoelectric focusing (IEF) has been applied to the study of the apolipoprotein components of rat serum high density and very low density lipoproteins. The apolipoproteins were separated on 7.5% polyacrylamide gels containing 6.8% urea, with a pH gradient of 4-6. The middle molecular weight range apolipoproteins were identified on IEF gels by the use of apolipoproteins purified by electrophoresis on gels containing sodium dodecyl sulfate (SDS). The A-1 protein focused as 4 to 5 bands from pH 5.46 to 5.82; the A-IV protein and the arginine-rich protein each focused as 4 to 6 bands from pH 5.31 to 5.46. The low molecular weight proteins focused from pH. 4.43 to 4.83 and are the subject of a separate communication. Comparisons of the IEF method with SDS gel electrophoresis, polyacrylamide gel electrophoresis in urea, and Sephadex chromatography are also reported. Additional studies were also carried out that tend to rule out carbamylation or incomplete unfolding of the proteins in the presence of urea as the causes of the observed heterogeneity.  相似文献   

3.
Using fast protein chromatofocusing, a high-efficiency column chromatography method with a self-generated pH gradient and focusing effects, soluble human very-low-density lipoprotein (VLDL) apolipoproteins were fractionated between pH 6.3 and 4.0. In the presence of 6 mol/l urea and with a flow rate of 1 ml/min, one run (up to 10 mg of protein) took 30 min. VLDL apolipoproteins were separated in seven peaks. As revealed by SDS-polyacrylamide gel electrophoresis, isoelectric focusing and double-immunodiffusion against mono-specific antisera, fractions corresponded to the following proteins: apolipoprotein C-I, albumin, apolipoproteins A-I, E, C-II plus C-III0, C-III1 and C-III2, respectively. Apolipoproteins were eluted in sharp, well-resolved peaks. The recovery of proteins was 78% of the starting material. With fast protein chromatofocusing, an efficient isolation of single apolipoproteins is possible from small amounts of VLDL apolipoprotein preparations. This technique is superior to the commonly used, time-consuming methods for apolipoprotein isolation.  相似文献   

4.
We have investigated the self-association properties of human apolipoprotein A-IV using several complementary physical techniques. Sedimentation equilibrium analysis demonstrated that human apolipoprotein A-IV formed oligomeric species in aqueous solution at physiologic pH. Computer analysis established that the best model of self-association is a monomer-dimer-tetramer scheme, with an unusually large monomer-dimer association constant of 2.9 X 10(5) liters/mol. Fluorescence spectroscopy and electrophoretic analysis demonstrated that the rate of monomer-oligomer interconversion is sufficiently slow that a stable population of dimeric protein exists in solution, even at low total protein concentrations, and that the extent of dimerization is minimally influenced by pH. Moreover, these techniques established that the dissociation of oligomeric forms and the unfolding of the monomeric form are discrete and sequential events. In experiments where apolipoprotein A-IV was incubated with human high density lipoproteins, fractionated by gradient gel electrophoresis, and localized by immunoblotting, dimer formation occurred, but very little binding to lipoproteins was observed. Immunoblots of human serum fractionated on acrylamide gradient gels and isopycnic density gradients demonstrated an apolipoprotein A-IV band of size and density consistent with a circulating dimeric form, unassociated with lipid. We conclude that human apolipoprotein A-IV undergoes high affinity self-association in aqueous solutions, and that such self-association likely occurs in vivo. Self-association may thus be important in determining the biologic behavior of human apolipoprotein A-IV by influencing both the kinetics and distribution of its association with plasma lipoproteins.  相似文献   

5.
A simple method for screening populations for the apolipoprotein E polymorphism which involves isoelectric focusing of delipidated samples on polyacrylamide gels of pH 4.5-5.8 followed by immunoblotting using a double-antibody technique is presented. The method is a synthesis of two previously published procedures and works well on samples that have been stored for as long as 15 years.  相似文献   

6.
Plasma lipoproteins from 5-week old male chickens were separated over the density range 1.006-1.172 g/ml into 22 subfractions by isopycnic density gradient ultracentrifugation, in order to establish the distribution of these particles and their constituent apolipoproteins as a function of density. Lipoprotein subfractions were characterized by electrophorectic, chemical and morphological analyses, and their protein moieties were defined according to net charge at alkaline pH, molecular weight and isoelectric point. These analyses have permitted us to reevaluate the density limits of the major chicken lipoprotein classes and to determine their main characteristics, which are as follows: (1) very-low-density lipoproteins (VLDL), isolated at d less than 1.016 g/ml, were present at low concentrations (less than 0.1 mg/ml) in fasted birds; their mean diameter determined by gradient gel electrophoresis and by electron microscopy was 20.5 and 31.4 nm respectively; (2) as the the density increased from VLDL to intermediate density lipoproteins (IDL), d 1.016-l.020 g/ml) and low-density lipoproteins (LDL, d 1.020-1.046 g/ml), the lipoprotein particles contained progressively less triacylglycerol and more protein, and their Stokes diameter decreased to 20.0 nm; (3) apolipoprotein B-100 was the major apolipoprotein in lipoproteins of d less than 1.046 g/ml, with an Mr of 350000; small amounts of apolipoprotein B-100 were detectable in HDL subfractions of d less than 1.076 g/ml; urea-soluble apolipoproteins were present in this density range as minor components of Mr 38000-39000, 27000-28000 (corresponding to apolipoprotein A-1) and Mr 11000-12000; (4) high density lipoprotein (HDL, d 1.052-1.130 g/ml) was isolated as a single band, whose protein content increased progressively with increase in density; the chemical composition of HDL resembled that of human HDL2, with apolipoprotein A-1 (M 27000-28000) as the major protein component, and a protein of Mr 11000-12000 as a minor component; (5) heterogeneity was observed in the particle size and apolipoprotein distribution of HDL subfractions: two lipoprotein bands which additional apolipoproteins of Mr 13000 and 15000 were detected. These studies illustrate the inadequacy in the chicken of the density limits applied to fractionate the lipoprotein spectrum, and particularly the inappropriateness of the 1.063 g/ml density limit as the cutoff for LDL and HDL particle populations in the species.  相似文献   

7.
Chromatofocusing, which separates proteins based on differences in isoelectric point, has been used on the Fast Protein Liquid Chromatography (FPLC) system (Pharmacia) to separate the C apolipoproteins from human very low density lipoproteins (VLDL). Using a Mono P column (Pharmacia), a pH gradient between pH 6.2 and pH 4.0 was generated using buffers containing 6 M urea, at a flow rate of 0.5 ml/min. Typically, runs took approximately 45 min. Chromatofocusing of delipidated whole VLDL produced sharp, well-resolved peaks for the C apolipoproteins. However, as determined by analytical isoelectric focusing (IEF), the apolipoprotein E isoforms were not separated from apoC-II, and they contaminated the other apoC species to a variable extent. In addition, apoC-II was not resolved from apoC-III0. Preliminary precipitation of VLDL with acetone prior to delipidation removed both apolipoproteins E and B. Using a start buffer of 25 mM histidine, pH 6.2, and a 1:30 dilution of the polybuffer exchanger (eluting buffer), apoC-II, C-III0, C-III1, and C-III2 were well resolved in run-times of approximately 60 min. The C apoproteins proved to be pure by analytical IEF and immunoassay with monospecific antisera against apoC-II and C-III. Recovery was over 90% of the protein chromatographed. In addition, a variant of apoC-II present in VLDL of a hypertriglyceridemic subject was clearly resolved from the other C apolipoproteins. This technique is superior to conventional methodology in terms of its time saving and high resolution. The application of this technique to the study of C apolipoprotein variants and C apolipoprotein specific radioactivity determinations is possible.  相似文献   

8.
PURPOSE OF REVIEW: Binding of apolipoprotein B-100-containing lipoproteins (VLDL, IDL, and LDL) to proteoglycans and modifications of the lipoproteins, whether bound or unbound, are key processes in atherogenesis. The complex interplay between binding and modification has been studied at neutral pH conditions. It has been demonstrated that during atherogenesis the extracellular pH of the lesions decreases. We summarize findings suggesting that lipoprotein binding and modification are enhanced at acidic pH. RECENT FINDINGS: Many enzymes found in the arterial intima, such as secretory sphingomyelinase and cathepsins, are able to hydrolyze lipoproteins in vitro. These enzymes function optimally at slightly acidic pH (pH 5.5-6.5), and are likely to act on lipoproteins optimally in the acidic plaque areas. Also, the ability of human aortic proteoglycans to bind native VLDL, IDL, and LDL is dramatically increased at acidic pH; this binding can be further increased if these apolipoprotein B-100-containing particles are hydrolytically modified. SUMMARY: Recent in-vitro findings suggest that in areas of atherosclerotic arterial intima where the extracellular pH is decreased, binding of apolipoprotein B-100-containing lipoproteins to proteoglycans and modification of the lipoproteins by acidic enzymes are enhanced. The pH-induced amplification of these processes will lead to enhanced extracellular accumulation of lipoproteins and accelerated progression of the disease.  相似文献   

9.
Plasma HDL-cholesterol and apolipoprotein A-I (apoA-I) levels are strongly inversely associated with cardiovascular disease. However, the structure and protein composition of HDL particles is complex, as native and synthetic discoidal and spherical HDL particles can have from two to five apoA-I molecules per particle. To fully understand structure-function relationships of HDL, a method is required that is capable of directly determining the number of apolipoprotein molecules in heterogeneous HDL particles. Chemical cross-linking followed by SDS polyacrylamide gradient gel electrophoresis has been previously used to determine apolipoprotein stoichiometry in HDL particles. However, this method yields ambiguous results due to effects of cross-linking on protein conformation and, subsequently, its migration pattern on the gel. Here, we describe a new method based on cross-linking chemistry followed by MALDI mass spectrometry that determines the absolute mass of the cross-linked complex, thereby correctly determining the number of apolipoprotein molecules in a given HDL particle. Using well-defined, homogeneous, reconstituted apoA-I-containing HDL, apoA-IV-containing HDL, as well as apoA-I/apoA-II-containing HDL, we have validated this method. The method has the capability to determine the molecular ratio and molecular composition of apolipoprotein molecules in complex reconstituted HDL particles.  相似文献   

10.
The purposes of this study were to establish the role of disulfide linkages in the secondary structure of apolipoprotein B, to investigate the effects of sulfhydryl blocking agents, denaturing agents, pH and storage on the conformation of apolipoprotein B and lipoprotein B, and to compare the conformation of water-soluble apolipoprotein B in the presence and absence of its lipids by using circular dichroism. Fresh lipoprotein B examined in Tris/EDTA at pH 9.0, 7.3 and 2.7 exhibited alpha-helical content of 24.4, 26.7 and 26.9%, and beta-pleated sheet 25.1, 15.4 and 18.0%, respectively. The carboxymethylated (CM-) lipoprotein B had similar alpha-helical contents, and lower contents of beta-sheets. Storage of lipoprotein B resulted in marked change of beta-sheets and gradual decrease in alpha-helical structure, in spite of the preventive measures taken for lipid peroxidation and proteolytic degradation. Exposure of apolipoprotein B to 6 M guanidine X HCl led to a complete disappearance of the alpha-helix with an increase in the beta-sheets to 35-40%, irrespective of the use of disulfide-reducing agents. By substituting 6 M urea for guanidine X HCl, the alpha-helical contents for both CM- and reduced CM-apolipoprotein B increased up to 7-9% with a concomitant decrease in beta-structure. When urea was replaced with aqueous buffers, these apolipoprotein B preparations regained their alpha-helical contents (25-27%) to the full extent originally present in the parent lipoprotein samples. No difference was observed between the secondary structure of CM- and reduced CM-apolipoprotein B. Furthermore, the conformation of apolipoprotein B did not vary with pH when pH was changed from 2.7 to 9.0. These results suggest that (1) the conformation of apolipoprotein B is more stable with respect to pH in the absence of lipids than in their presence, (2) intramolecular disulfide linkages play an insignificant role in the conformation of apolipoprotein B, and (3) the changes in alpha-helix structure of lipoprotein B or CM-lipoprotein B due to delipidization and denaturation are reversible.  相似文献   

11.
H Hauser  H H Mantsch  H L Casal 《Biochemistry》1990,29(9):2321-2329
31P NMR and infrared spectroscopic methods have been used to study the formation of small unilamellar vesicles by the pH-jump method. It is shown that increasing the pH of different lamellar phospholipid dispersions (phosphatidic acids and phosphatidylserines) induces a pH gradient. This pH gradient is estimated to be 4 +/- 1 pH units, and its direction is such that the inner monolayer of the vesicles is at lower pH. There is spectroscopic evidence for tighter packing of the lipid hydrocarbon chains in the inner monolayer, probably due to the constraints imposed by the high curvature of the small vesicles formed. These results are discussed in terms of the driving force of the spontaneous vesiculation.  相似文献   

12.
To determine the concentration and distribution of apolipoproteins C-III and E in low density lipoproteins (LDL) of d 1.025-1.043 g/ml, fresh human plasma was fractionated by single-spin density gradient ultracentrifugation into five layers. Two major subpopulations including layer 2 (d 1.025-1.029 g/ml) and layer 3 (d 1.032-1.043 g/ml) were isolated and characterized by determination of flotation coefficient, neutral lipids and apolipoproteins B, C-III and E. The apolipoprotein B/C-III/E ratio of layer 2 was 100/(3.3 +/- 2.0)/(5.1 +/- 2.9) (wt/wt) and that of layer 3 was 100/(0.61 +/- 0.32)/(0.58 +/- 0.29) (wt/wt). These weight ratios corresponded to molar ratios of 1.0/(1.90 +/- 1.16)/(0.74 +/- 0.42) and 1.0/(0.34 +/- 0.18)/(0.08 +/- 0.04), respectively. Layer 2 contained 6-23% of the total plasma apolipoprotein B or 7-27% of total LDL2 (d 1.019-1.063 g/ml) apolipoprotein B. Layer 3 contained 41-65% of plasma apolipoprotein B or 62-86% of LDL2 apolipoprotein B. About 5-17% of apolipoprotein C-III and 8-30% of apolipoprotein E in plasma are distributed in layers 2 and 3 with the majority present in layer 2. These results show an evident apolipoprotein heterogeneity of LDL2 isolated from normolipidemic subjects. Moreover, they show that the relatively small amounts of apolipoprotein C-III and apolipoprotein E in lower-density segments of LDL2 take on a greater significance when presented in molar rather than weight concentrations. The existence of different ratios of apolipoprotein C-III/apolipoprotein E in layer 2 and layer 3 suggest the presence in LDL2 of varying amounts of several discrete apolipoprotein B- and/or apolipoprotein C-III- and apolipoprotein E-containing lipoprotein particles.  相似文献   

13.
Procedures for the isolation of two lipoprotein fractions from plasma high-density lipoproteins (HDL), characterized by apolipoprotein A-I and apolipoprotein A-I together with apolipoprotein A-II, have been elaborated. Apolipoprotein A-I was identified as the protein moiety of one of these fractions (lipoprotein A-I) with polyacrylamide gel electrophoresis (at basic and acidic pH, as well as in the presence of sodium dodecyl sulphate), immuno-double-diffusion, and amino acid analysis. Apolipoproteins A-I and A-II were identified as the protein moiety of the other fraction (lipoprotein A) with polyacrylamide gel electrophoresis (basic and acidic pH) and immuno-double-diffusion. Lipoprotein A-I consisted of spherical particles with a diameter similar to that of HDL as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 8.7 nm from gel chromatography. Lipoprotein A-I migrated in the HDL position on crossed immunoelectrophoresis. On iso-electric focusing lipoprotein A-I appeared as multiple bands in the pH range 5.05-5.55. Lipoprotein A-I had the density of an HDL-2 fraction (rho: 1.063-1.105). Lipoprotein A consisted of spherical particles with a diameter similar to that of HDL, as judged from negative strains in the transmission electron microscope. The diameter was estimated to be 7.9 nm from gel chromatography. The molar ratio between the A-I and A-II polypeptides was estimated to 1.3:1 with electroimmunoassay and calculations from the amino acid compositions. Lipoprotein A migrated in the position of HDL on crossed immuno-electrophoresis. On iso-electric focusing lipoprotein A appeared as one major and two minor bands in the pH range 5.10-5.30. Lipoprotein A had the hydrated density of an HDL-2 fraction.  相似文献   

14.
Three mouse monoclonal antibodies (Mabs) to human apo A-I were produced using apolipoprotein A-I or HDL3 as immunogens. These monoclonal antibodies, 2G11, 4A12 and 4B11, were characterized for their reactivity with isolated apolipoprotein A-I and HDL in solution. The immunoblotting patterns of the HDL3 two-dimensional electrophoresis show that these three monoclonal antibodies reacted with all the polymorphic forms of apolipoprotein A-I. Cotitration experiments indicated that they correspond to three distinct epitopes. In order to locate these three antigenic determinants on the isolated apolipoprotein A-I, the reactivity of the three monoclonal antibodies has been studied on CNBr-cleaved apolipoprotein A-I. The monoclonal antibodies 2G11 and 4A12 addressed to the amino (CNBr 1) and carboxy (CNBr 4) terminal segments, respectively. In comparison with the monoclonal antibodies characterized by Weech et al. ((1985) Biochim. Biophys. Acta 835, 390-401), monoclonal antibody 4A12 is the only one described in the literature which is specific of the carboxy terminal segment of apolipoprotein A-I. Monoclonal antibody 4B11 does not react with any CNBr fragment, its binding is temperature dependent, it could be directed to a conformational epitope. Relative differences were demonstrated in the expression of the three epitopes in HDL subfractions isolated by density gradient ultracentrifugation. According to Curtiss and Edgington ((1985) J. Biol. Chem. 260, 2982-2993) our results indicate the existence of an immunochemical heterogeneity in the organization of apolipoprotein A-I at the surface of HDL particles as well as in the soluble form of apolipoprotein A-I.  相似文献   

15.
The feasibility of purifying subcellular membranes, especially plasma membranes, from oat roots using isoelectric focusing has been examined. Membranes from oat (Avena sativa L. cv Garry) root homogenates were fractionated using discontinuous sucrose density gradient centrifugation and then electrofocused using a microanalytical isoelectric focusing column. The column contained either a broad-range (pH 3-10) or narrow-range (pH 3-6) pH gradient stabilized by a 5 to 15% Ficoll gradient. Results from the broad-range columns confirmed that the isoelectric pH (pI) values of the membranes were in the acidic range, with pI values ranging from 3.9 to 5.2. Using narrow-range pH gradients, it was possible to fractionate further plasma membrane-enriched material obtained from a sucrose density gradient. We had no success at fractionating crude membrane preparations from oat roots. Narrow-range pH gradients generated by commercial ampholytes were more successful than those generated by acetate/acetic acid mixtures.  相似文献   

16.
Beta2-glycoprotein I has a high affinity for triglyceride-rich particles, activates lipoprotein lipase, and is also defined as an apolipoprotein H. Previous studies have shown that apolipoprotein H is a regular structural component of the major classes of lipoproteins. In view of these findings, we analyzed the interactions of apolipoprotein H with lipoproteins in the fasting plasma of eight normal, seven hypertriglyceridemic, and seven hypercholesterolemic subjects. After rate-zonal, density gradient ultracentrifugation, apolipoprotein H was little distributed among the different density fractions, and most of it was recovered in the last fraction that contained the lipoprotein-free plasma. A small percentage (4-13%) of the apolipoprotein H associated with plasma lipoproteins was detected at the density ranging from 1.090 to 1.225 g/ml. This result means that apolipoprotein H is little associated with lipoproteins.  相似文献   

17.
Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.  相似文献   

18.
A new acrylamido buffer has been synthesized, for use in isoelectric focusing in immobilized pH gradients. This compound (2-acrylamido glycolic acid) has a pK = 3.1 (at 25 degrees C, 20 mM concentration during titration) and is used, by titration with the pK 9.3 Immobiline, to produce a linear pH gradient in the pH 2.5-3.5 interval. Pepsin (from pig stomach) focused in this acidic pH gradient is resolved into four components, two major (with pI values 2.76 and 2.78) and two minor (having pI values 2.89 and 2.90). This is the first time that such strongly acidic proteins could be focused in an immobilized pH gradient. Even in conventional isoelectric focusing in amphoteric buffers it has been impossible to focus reproducibly very-low-pI macromolecules.  相似文献   

19.
A new method of producing a stable pH gradient in buffer solutions is suggested, obtained by the concentration gradient of a nonelectrolyte in buffer solutions as a result of the gradual change in the dielectric properties of the solution. The maximal concentrations of nonelectrolyte which do not influence the protein configuration allow a pH gradient with a range of two pH units to be produced. It is suggested that the properties of some polyols (i.e. glycerol or mannitol) be used to change the pH of the borate buffer for the production of greater pH gradient with a range of up to 4-5 pH units. Creating the gradient of concentration of polyols, one can obtain a pH gradient in borate buffer solutions. Though the polylydroxyl compound-borate complexes posses mobility in an electric field, a stable pH gradient can be achieved during 12 days of electrophoresis. The isoelectric focusing of haemoglobin, human serum albumin and immunoglobulins was carried out in both systems suggested. These findings were compared with isoelectric focusing in Ampholines. There was a good agreement between the methods compared. The possible differences are discussed.  相似文献   

20.
Ye XY  Ng TB 《Life sciences》2000,67(8):887-894
A new whey protein designated apolipoprotein H-like whey protein, with a molecular weight of 62 kDa and an N-terminal amino acid sequence similar to that of apolipoprotein H, was isolated from bovine milk. The isolation procedure involved removal of globulin from acid whey by precipitation with 1.8M (NH4)2SO4, followed by addition of (NH4)2SO4 to attain a concentration of 3.6M. Subsequent steps included chromatography on CM-Sepharose and Mono S and elution of the adsorbed protein of interest with a linear NaCl gradient. The new whey protein displayed some ribonuclease (RNase) activity. It was most active at pH 7.5 with yeast transfer RNA (tRNA) as substrate and showed potent specific ribonucleolytic activity toward poly C. It inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of approximately 63 nM.  相似文献   

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